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WO1997047744A2 - T-cell selective interleukin-4 agonists - Google Patents

T-cell selective interleukin-4 agonists Download PDF

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Publication number
WO1997047744A2
WO1997047744A2 PCT/US1997/009286 US9709286W WO9747744A2 WO 1997047744 A2 WO1997047744 A2 WO 1997047744A2 US 9709286 W US9709286 W US 9709286W WO 9747744 A2 WO9747744 A2 WO 9747744A2
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WIPO (PCT)
Prior art keywords
leu
mutein
human
thr
substituted
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PCT/US1997/009286
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English (en)
French (fr)
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WO1997047744A3 (en
Inventor
Armen B. Shanafelt
Jeffrey Greve
Robert Gundel
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Bayer Corporation
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Priority to PL97330413A priority Critical patent/PL187846B1/pl
Priority to AU32220/97A priority patent/AU725090B2/en
Priority to CA002256459A priority patent/CA2256459A1/en
Priority to EP97927864A priority patent/EP0912741B1/en
Priority to AT97927864T priority patent/ATE272118T1/de
Priority to JP10501642A priority patent/JP2000515007A/ja
Priority to DE69730029T priority patent/DE69730029T2/de
Priority to IL12699597A priority patent/IL126995A0/xx
Priority to NZ332790A priority patent/NZ332790A/xx
Priority to BR9709715A priority patent/BR9709715A/pt
Publication of WO1997047744A2 publication Critical patent/WO1997047744A2/en
Publication of WO1997047744A3 publication Critical patent/WO1997047744A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5406—IL-4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P19/00—Drugs for skeletal disorders
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention is generally related to the fields of pharmacology and immunology.
  • the invention is directed to novel compositions of matter for selectively activating T cells, and having reduced activation of Endothelial cells or fibroblasts.
  • novel compositions include variants of the cytokine family, and in particular human
  • Interleukin-4 (IL-4).
  • Interleukin 4 is a pleiotropic cytokine, having activities on cells of the immune system, endothelium, and those of fibroblastic nature. Reported in vitro effects of IL-4 administration include proliferation of B cells, immunoglobulin class switching in B cells. In T cells, IL-4 stimulates T cell proliferation after preactivation with mitogens and down-regulates IFN- ⁇ production. In monocytes, IL-4 induces class II MHC molecules expression, release of lipopolysaccharide-induced tPA, and CD23 expression. In Endothelial cells (EC), IL4 induces expression of VCAM-1 and IL-6 release, and decreases ICAM-1 expression. (Maher, DW, et al. Human Interleukin-4: An Immunomodulator with Potential Therapeutic Applications, Progress in Growth Factor Research, 3:43-56 (1991)). Because of its ability to stimulate proliferation of T cells activated by exposure to
  • IL-4 has been pursued.
  • IL-4 has demonstrated anti-neoplastic activity in animal models of renal carcinomas, and has induced tumor regression in mice (Bosco, M., et al, Low Doses of IL-4 Injected Perilymphatically in Tumor-bearing Mice Inhibit the Growth of Poorly and Apparently Nonimmunogenic Tumors and Induce a Tumor Specific Immune Memory, J. Immunol.. 145:3136-43 (1990)).
  • its toxicity limits dosage in humans (Margolin, K, et al, Phase II Studies of Human Recombinant Interleukin-4 in Advanced Renal Cancer and Malignant Melanoma, I Immunotherapy. 15:147-153 (1994)).
  • Th cells fall into two broad classes, designated Thl and Th2 (Mosmann, T.R., Cherwinski, H, Bond, M. W., Giedlin, M.A. and Coffman, R.L., Two types of murine helper T cell clone.
  • Thl and Th2 Two broad classes of murine helper T cell clone.
  • T cell classes are defined by the cytokines they express: Thl cells make IL-2, INF- ⁇ , and TNF- ⁇ , while Th2 cells make IL-4 and IL-5. Thl and Th2 cells are formed from naive CD4+ T cells. Differentiation into Thl or Th2 subsets depends on the cytokine present during antigen stimulation: IFN- ⁇ and IL-12 direct differentiation of naive cells to the Thl phenotype, while IL-4 directs differentiation to the Th2 phenotype.
  • Thl and Th2 subsets may represent extremes along a continuum of Th cell phenotypes (for example, ThO cells, which express low levels of both INF- ⁇ and IL-4, have been described), this classification nevertheless is the major paradigm in the field of immunology for describing the character of the immune response.
  • autoimmune diseases are associated with a predominantly Thl T cell response against autoantigen (Liblau RS; Singer SM; McDevitt HO, Thl and Th2 CD4 * T cells in the pathogenesis of organ- specific autoimmune diseases, Immunol. Today. 16:34-38 (1995)).
  • IDDM insulin-dependent diabetes
  • Thl -type cells are primarily responsible for the pancreatic ⁇ cell destruction (reviewed in Tisch, R. et al, Review: Insulin-dependent Diabetes Mellitus, Cell. 85:291-297 (1996)).
  • IL-4 IL-4 Reverses T cell Proliferation Unresponsiveness and Prevents the Onset of Diabetes in NOD Mice, J. Exp. Med.. 178:87-99 (1993)
  • Another such autoimmune disease is multiple sclerosis (MS), a disease which is characterized by an autoimmune attack upon the myelin sheath surrounding nerve cells.
  • autoimmune diseases such as Rheumatoid Arthritis (RA) are also targets for IL-4 based therapies.
  • Animal models of RA have shown a disequilibrium of cell profiles tilting towards Thl cells, and in mice that overexpress TNF- ⁇ , anti-TNF- ⁇ antibodies have demonstrated disease attenuation, suggesting that IL-4 therapies that result in down- regulation of Thl cell populations may have an anti-TNF- ⁇ effect also. (See Feldmann, M., et al., Review: Rheumatoid Arthritis, Cel ⁇ , 85:307-310 (1996)).
  • Psoriasis vulgaris is a chronic dermatologic disorder characterized by infiltration of affected skin with monocytes and T cells.
  • monocytes and T cells Several reports indicate that psoriatic skin lesional T cells and PBL are predominantly of the Thl phenotype (Uyemura K; Yamamura M; Fivenson DF; Modlin RL; Nickoloff BJ, The cytokine network in lesional and lesion-free psoriatic skin is characterized by a T-helper type 1 cell-mediated response, J Invest Dermatol..
  • IL-4 would be expected to reverse the Th polarization and be of clinical benefit in psoriasis. Certain infectious diseases are associated with polarized Th cell responses to the infectious agent.
  • Th2 responses have in some cases been associated with resistance to the infectious agent.
  • An example is Borrelia burgdorfei, the infectious agent for Lyme disease. Humans infected with B. burgdorferi exhibit a predominantly Thl -like cytokine profile (Oksi J; Savolainen J; Pene J; Bousquet J; Laippala P; Viljanen MK, Decreased interleukin-4 and increased gamma interferon production by peripheral blood mononuclear cells of patients with Lyme borreliosis, Infect. Immun.. 64:3620-3623 (1996)). In a mouse model of B.
  • burgdorferi-intecled mice with IL-4 augments resistance to the infection (Keane-Myers A; Maliszewski CR; Finkelman FD; Nickell SP, Recombinant IL-4 treatment augments resistance to Borrelia burgdorferi infections in both normal susceptible and antibody-deficient susceptible mice, _J Immunol.. 156:2488-2494(1996)).
  • IL-4 has been reported to have a direct effect on inhibiting the growth of lymphomas and leukemias (Akashi, K, The role of interIeukin-4 in the negative regulation of leukemia cell growth, Leuk Lvmphoma. 9:205-9 ( 1993)).
  • IL-4 has been reported to induce apoptosis in cells from patients with acute lymphoblastic leukemia (Manabe, A, et al, Interleukin-4 induces programmed cell death (apoptosis) in cases of high-risk acute lymphoblastic leukemia, Blood.
  • osteoarthritis is a disease in which the degradation of cartilage is the primary pathology (Sack, KE, Osteoarthritis, A continuing challenge, West J Med. 163:579-86 (1995); Oddis, CV, New perspectives on osteoarthritis, Am J Med. 100:1 OS- 15S (1996)).
  • IL-4 inhibits TNF- ⁇ and IL-1 beta production by monocytes and synoviocytes from osteoarthritic patients (Bendrups, A, Hilton, A, Meager, A and Hamilton, JA, Reduction of tumor necrosis factor alpha and interleukin- 1 beta levels in human synovial tissue by interleukin-4 and glucocorticoid, Rheumatol Int. 12:217-20 (1993); Seitz, M, et al, Production of interleukin- 1 receptor antagonist, inflammatory chemotactic proteins, and prostaglandin E by rheumatoid and osteoarthritic synoviocytes- -regulation by IFN-gamma and IL-4, J Immunol.
  • IL-4 has been reported to directly block the degradation of cartilage in ex vivo cartilage explants (Yeh, LA, Augustine, AJ, Lee, P, Riviere, LR and Sheldon, A, Interleukin-4, an inhibitor of cartilage breakdown in bovine articular cartilage explants, J Rheumatol. 22: 1740-6 (1995)). These activities suggest that IL-4 would be of clinical benefit in osteoarthritis.
  • IL-4 has been limited due to its acute toxicity, which is manifested as a vascular leak syndrome (Margolin, K, el al, Phase II Studies of Human Recombinant Interleukin-4 in Advanced Renal Cancer and Malignant Melanoma, J. Immunotherapy. 15: 147- 153 (1994)).
  • vascular leak syndrome Margolin, K, el al, Phase II Studies of Human Recombinant Interleukin-4 in Advanced Renal Cancer and Malignant Melanoma, J. Immunotherapy. 15: 147- 153 (1994)
  • IL-4 mutant proteins are known.
  • the IL-4 mutein IL-4/Y124D is a T cell antagonist (Kruse N, Tony HP, Sebald IV, Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement, Embo J. 11:3237-44 (1992)).
  • IL-4 uses include the following: the use of IL-4 for potentiation of anticancer effects of chemotherapeutic agents, particularly Hodgkin's Disease and non-Hodgkins Lymphoma (see WO 9607422); the use of antigenic fragments of IL-4 to generate antibodies to treat IL-4 related diseases by suppressing or imitating the binding activity of IL-4 (see WO 9524481), and to detect, measure and immunopurify IL-4 (see WO 9317106); for inducing the differentiation of precursor B cells to Immunoglobulin secreting cells, the mature B cells being useful for restoring immune function in immune-compromised patients (see WO 9404658); when used in combination with IL-10, as a therapy for treatment of leukemia, lymphoma, inflammatory bowel disease and delayed type hypersensitivity (e.g.
  • ulcerative colitis and Crohn's Discase (see WO 9404180); treatment of HIV infection by administering IL-4 to inhibit viral replication in monocytes and macrophages, and to increase their cytotoxicity towards some tumor cells (see WO 9404179); for stimulation of skin fibroblast proliferation for treating wounds in diabetic and immuno-compromised patients (see WO 921 1861); for enhancing the primary immune response when administering bacterial, toxoid, and viral vaccines, especially tetanus toxoid vaccine (see WO 9211030); for inhibition of IL-2 induced proliferation of B cell malignancies, especially chronic lymphocytic leukemia, non-Hodgkin's malignant lymphoma (see WO 9210201); use of IL-4 to treat melanomas, renal and basal cell carcinomas (see WO 9204044).
  • the patent literature discloses IL-4 proteins and some muteins, but none directed to an IL-4 therapy with reduced side effects.
  • Lee et al. U.S. Pat. No. 5,017,691 (“the '691 patent") is directed to mammalian proteins and muteins of human IL-4 which disclose both B-cell growth factor activity and T cell growth factor activity. It discloses nucleic acids coding for polypeptides exhibiting IL-4 activity, as well as the polypeptides themselves and methods for their production. Muteins to the wild-type IL-4 at amino acid positions are disclosed that retain their ability to stimulate both B- and T cell proliferation in vitro.
  • IL-4 itself is not enabling as a therapeutic modality because of the dose- limiting toxicity.
  • U.S. Patent No. 5,013,824 describes hIL-4 peptide derivatives comprising from 6 to 40 amino acids of the native hIL-4.
  • immunogens comprising conjugates of the peptides and carriers. Carriers include erythrocytes, bacteriophages, proteins, synthetic particles or any substance capable of eliciting antibody production against the conjugated peptide. No muteins of IL-4 are disclosed.
  • WO96/04306-A2 discloses single-muteins that are antagonists and partial agonists of hIL-2 and hIL-13. No data regarding IL-4 is disclosed.
  • WO95/27052 discloses splice mutants of IL-2 and IL-4 containing exons 1, 2 and 4.
  • the invention is directed to human IL-4 muteins numbered in accordance with wild-type IL-4 having T cell activating activity, but having reduced endothelial cell activating activity.
  • human IL-4 muteins wherein the surface-exposed residues of the D helix of the wild-type IL-4 are mutated whereby the resulting mutein causes T cell proliferation, and causes reduced IL-6 secretion from HUVECs, relative to wild-type IL-4.
  • This invention realizes a less toxic IL-4 mutein that allows greater therapeutic use of this interleukin.
  • the invention also includes polynucleotides coding for the muteins of the invention, vectors containing the polynucleotides, transformed host cells, pharmaceutical compositions comprising the muteins, and therapeutic methods of treatment.
  • the invention is also directed to a vector comprising the polynucleotide encoding a mutein of this invention, the vector directing the expression of a human IL-4 mutein having T cell activating activity but having reduced endothelial cell activating activity, the vector being capable of enabling transfection of a target organism and subsequent in vivo expression of said human IL-4 mutein coded for by said polynucleotide.
  • the invention is also directed to a method of selecting a human IL-4 mutein numbered in accordance with wild-type IL-4 having T cell activating activity but having reduced endothelial cell activating activity, comprising mutating the surface-exposed residues of the D helix of the wild-type IL-4 whereby the resulting mutein causes T cell proliferation, and causes reduced IL-6 secretion from HUVECs, relative to wild-type.
  • the invention is also directed to a method of treating a patient afflicted with an IL-4 treatable condition by administering a therapeutically effective amount of a human IL-4 mutein numbered in accordance with wild-type IL-4 having T cell activating activity but having reduced endothelial cell activating activity.
  • Fig. 1 is an amino acid sequence (SEQ ID NO:l) of mature wild-type human IL-4 used in this study. Helices are underlined and labeled sequentially A, B, C, and D. Positions that, when mutated yielded cell-selective IL-4 agonists, are indicated in bold type.
  • Fig. 2 is a graphical presentation of the T cell selective agonist concept.
  • Fig. 3 is a composite dose response curve for selective agonist muteins in the HUVEC 1L-6 secretion assay.
  • Panel A O, wild-type IL-4; #, R121E;V, R121P;T, R121T/E122F/Y124Q.
  • Panel B O, wild-type IL-4; •, Y124Q; V, Y124R; T, Y124A/S125A.
  • Fig. 4 are individual dose response curves of selective agonist muteins in the HUVEC IL-6 secretion assay.
  • Panel A O, wild-type IL-4; •, R121E.
  • Panel B O, wild-type IL-4; #, R121 P.
  • Panel C O, wild-type IL-4; •, Y124Q.
  • Panel D O, wild-type IL-4; •, Y124R.
  • Panel E O, wild-type IL-4; •, YI24A/S125A.
  • Panel F O, wild-type IL-4; #, R121T/E122F/Y124Q.
  • Fig. 5 is a composite dose-response curve for selective agonist muteins for the biological response of IL-4 muteins in 1° T cell proliferation assays.
  • Panel A O, wild-type IL-4; •, R121E; V, R121P; T, R121T/E122F/Y124Q.
  • Panel B O, wild-type IL-4; •, Y124Q; V, Y124R; T, Y124A/S125A.
  • Fig. 6 are individual dose response curves of the 1° T cell proliferation assay.
  • Panel A O, wild-type IL-4; •, R121E.
  • Panel B O, wild-type IL-4; •, R121P.
  • Fig. 7 are individual dose response curves showing the antagonism of IL-4-induced IL-6 secretion on HUVEC by the T cell-selective agonist IL-4 muteins R121E (•) and Y124Q (V).
  • the dose response of the IL-4 antagonist R121 D/Y124D (O) is included as a control.
  • Fig. 9A are the individual dose response curves for IL-4 (O) and the T cell-selective agonist muteins R121E ( ⁇ ) and T13D/R121E (A) in the 1° T cell proliferation assay.
  • Fig. 9A are the individual dose response curves for IL-4 (O) and the T cell-selective agonist muteins R121E ( ⁇ ) and T13D/R121E (A) in the 1° T cell proliferation assay.
  • 9B are individual dose response curves showing the antagonism of IL-4-induced IL-6 secretion on HUVEC by the T cell-selective IL-4 agonist muteins R121E ( ⁇ ) and T13D/R121E (A).
  • IL-4 has been shown to mediate a variety of cellular responses in vitro, including various effects on B cells, T cells, and monocytes, as well as endothelial cells (Maher DW, Davis I, Boyd AW, Morstyn G: Human interleukin-4: an immunomodulator with potential therapeutic applications. Prog Growth Factor Res 3:43-56, 1991 ; Powrie F, Coffman RL: Cytokine regulation of T cell function: potential for therapeutic intervention. Immunol Today 14:270-4, 1993).
  • VCAM-l vascular cell adhesion molecule- 1
  • IL-6 Colotta F, Sironi M, Borre A, Luini W, Maddalena F, Mantovani A: Interleukin 4 amplifies monocyte chemotactic protein and interleukin 6 production by endothelial cells.
  • Tumor necrosis factor combines with IL- 4 or IFN-gamma to selectively enhance endothelial cell adhesiveness for T cells.
  • the IL-4 mutein IL-4/Y124D (substitution of Aspartic acid for Tyrosine at position 124) is a T cell antagonist (Kruse N, Tony HP, Sebald W: Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement. Embo J 11:3237-44, 1992). In vivo experiments performed by the inventors have demonstrated that IL-4/Y124D exhibits acute toxicity similar to that of wild-type 1L-4 in monkeys, a previously undescribed observation.
  • the cellular events associated with both wild-type IL-4- and IL-4/Y124D-mediated toxicity include upregulation of VCAM-1, upregulation of MCP-1 in serum, increases in circulating monocytes together with a concomitant decrease in circulating lymphocytes, and an increase in hematocrit. Similar cellular trafficking has been observed in clinical trials using IL-4 in humans (Wong HL, Lotze MT, Wahl LM, Wahl SM: Administration of recombinant IL-4 to humans regulates gene expression, phenotype, and function in circulating monocytes. J Immunol 148:2118-25, 1992).
  • IL-4/Y124D Due to its properties as an antagonist of T cells, these results suggest that the toxicities demonstrated by IL-4/Y124D are due to agonist activities on and are mediated through cells other than T cells.
  • the observed in vivo toxicities using IL-4/Y124D and the known effects of IL-4 on endothelial cells are consistent with the mechanism that in vivo IL-4 toxicity is mediated through direct effects of IL-4 on the vascular endothelium.
  • IL-4 receptor may exist on endothelial cells ("EC"). This possibility led to efforts to synthesize IL-4 muteins that would selectively activate T cells, but not EC. While T cells express an IL-4 receptor composed of IL-4R ⁇ and IL-2R ⁇ subunits, the inventors have discovered that human umbilical vein endothelial cells (HUVEC), express IL-4R ⁇ but not IL-2R ⁇ . Crosslinking studies have shown that two receptor chains are expressed at the cell surface of HUVEC: the molecular weight of one is consistent with IL-4R ⁇ , and a second,. lower molecular weight chain.
  • HUVEC human umbilical vein endothelial cells
  • Figure 2 demonstrates graphically the selective agonist concept. It shows the T cell IL-4 receptor comprising the IL-4R ⁇ /IL-2R ⁇ subunit, and an Endothelial cell IL-4 receptor comprising the IL-4R ⁇ / ⁇ -like receptor subunit. Although depicted here together for purposes of illustration only, the two receptors for IL-4 are expressed on different cell types.
  • the T cell receptor is composed of IL-4R ⁇ and IL-2R ⁇ ; IL-4 binding induces receptor heterodimer formation that results in cellular signalling.
  • IL-4 induced receptor heterodimer formation occurs in a similar manner on EC's, except that the receptor for IL-4 is composed of IL-4R ⁇ and a y-like receptor component.
  • T cell-selective IL-4 agonists are those variants of IL-4 that retain their ability to interact with the T cell receptor IL-4R ⁇ /IL-2R ⁇ , but are unable to induce heterodimerization, and thus signalling, of the non-T cell receptor IL- 4Ra/y-like subunit.
  • Such T cell-selective IL-4 agonists retain their ability to interact with IL-4R ⁇ ; it is their ability to discriminate between IL-2R ⁇ and the y-like subunit that gives them their cell-selective activation properties.
  • the two components of the T cell receptor, IL4-R ⁇ and IL-2R ⁇ contact different regions of the IL-4 molecule, and therefor the inventors have focussed on a small region of IL-4 to modify. Hypothesizing that the novel receptor subunit would contact the same region of IL4 as does IL-2R ⁇ , the inventors made a number of substitutions in the D- Helix, particularly residues 121, 124 and 125.
  • the D-helix has been implicated in interactions with both IL-2R ⁇ and with the putative novel receptor on HUVEC (specifically, the IL-4 mutein R121D/Y124D is a HUVEC antagonist).
  • Muteins containing modifications to the D-helix of IL-4 were screened for their ability to stimulate either T cell proliferation or human umbilical vein endothelial cell (HUVEC) secretion of IL-6.
  • Muteins that induced a differential response on T cells relative to HUVEC were further characterized through further mutagenesis.
  • IL-4 Specific substitutions derived from an alignment between IL-2 and IL-4 were introduced into IL-4. These included: Arg-1 15 to Phe; Lys-1 17 to Asn; Glu-122 to Phe; Lys-126 to He; and three simultaneous changes Arg-121 to Thr, Glu-122 to Phe, and Tyr- 124 to Gin.
  • Mutations were introduced using site-directed mutagenesis on wild-type human IL-4 cDNA. Correct clones were subcloned to an expression vector suitable for expression in a heterologous system (e.g., E. coli, baculovirus, or CHO cells). Purified proteins were tested in T cell proliferation and HUVEC cytokine secretion assays (IL-6). Different responses generated by individual muteins between these assays, either in EC50 or maximal response (plateau) indicate mutations that effect these activities. Specifically, muteins that stimulate a relatively stronger response in the T cell assay (vs. wild-type IL-4) as compared to the response on HUVEC (vs.
  • wild-type IL-4 will suggest positions that are more important to the interaction of IL-4 with IL-2R ⁇ than the interaction of IL-4 with the novel HUVEC 1L-4 receptor. Further analysis and mutagenesis (e.g. combinatorial changes, substitution with all amino acids) of the identified positions will produce an IL-4 mutein with selective agonist properties for the T cell IL-4 receptor. This protein will also be a selective antagonist for IL-4-induced HUVEC responses.
  • wild type IL-4" means IL-4, whether native or recombinant, having the 129 normally occurring amino acid sequence of native human IL-4, as shown, e.g., in Figure 1.
  • IL-4 mutein means a polypeptide wherein specific substitutions to the human mature interleukin-4 protein have been made.
  • the arginine residue (R) at position 121 when numbered in accordance with wild type IL-4, is substituted with alanine (A), aspartate (D), glutamate (E), phenylalanine (F), histidine (H), isoleucine (I), lysine (K), asparagine (N) proline (P), threonine (T) or tryptophan (W); or the glutamate (E) residue at position 122 is substituted with phenylalanine (F); or the tyrosine residue at position 124 is substituted with alanine (A), glutamine (Q), arginine (R) serine (S) or threonine (T); or the serine (S) residue at position 125 is substituted with alanine (A).
  • IL-4 muteins have an amino acid sequence identical to wild type IL-4 at the other, non-substituted residues.
  • the IL-4 muteins of this invention may also be characterized by amino acid insertions, deletions, substitutions and modifications at one or more sites in or at the other residues of the native IL-4 polypeptide chain.
  • any such insertions, deletions, substitutions and modifications result in an IL-4 mutein that retains a T cell-selective activity while having reduced ability to activate endothelial cells.
  • IL-4 is known to have six cys residues, at wild-type positions 3, 24, 46, 65, 99 and 127.
  • numbered in accordance with wild type IL-4" we mean identifying a chosen amino acid with reference to the position at which that amino acid normally occurs in wild type IL-4.
  • the ser (S) normally occuring at position 125, when numbered in accordance with wild type IL-4, may be shifted in position in the mutein.
  • the location of the shifted ser (S) can be readily determined by inspection and correlation of the flanking amino acids with those flanking ser in wild type IL-4.
  • the IL-4 muteins of the present invention can be produced by any suitable method known in the art. Such methods include constructing a DNA sequence encoding the IL-4 muteins of this invention and expressing those sequences in a suitably transformed host. This method will produce recombinant muteins of this invention. However, the muteins of this invention may also be produced, albeit less preferably, by chemical synthesis or a combination of chemical synthesis and recombinant DNA technology.
  • a DNA sequence is constructed by isolating or synthesizing a DNA sequence encoding the wild type IL-4 and then changing the codon for arg!21 to a codon for alanine (A), aspartate (D), glutamate (E), phenylalanine (F), histidine (H), isoleucine (I), lysine (K), asparagine (N) proline (P), threonine (T) or tryptophan (W) by site-specific mutagenesis.
  • A alanine
  • D aspartate
  • E glutamate
  • F phenylalanine
  • H histidine
  • K lysine
  • K asparagine
  • N proline
  • T threonine
  • W tryptophan
  • a gene which encodes the desired IL-4 mutein may be synthesized by chemical means using an oligonucleotide synthesizer.
  • Such oligonucleotides are designed based on the amino acid sequence of the desired IL-4 mutein, and preferably selecting those codons that are favored in the host cell in which the recombinant mutein will be produced.
  • the genetic code is degenerate — that an amino acid may be coded for by more than one codon.
  • Trp (F) is coded for by two codons, TTC or TTT
  • tyr (Y) is coded for by TAC or TAT
  • his (H) is coded for by CAC or CAT.
  • Trp (W) is coded for by a single codon, TGG.
  • the DNA sequence encoding the IL-4 mutein of this invention may or may not also include DNA sequences that encode a signal sequence.
  • Such signal sequence should be one recognized by the cell chosen for expression of the IL-4 mutein. It may be prokaryotic, eukaryotic or a combination of the two. It may also be the signal sequence of native IL-4. The inclusion of a signal sequence depends on whether it is desired to secrete the IL-4 mutein from the recombinant cells in which it is made. If the chosen cells are prokaryotic, it generally is preferred that the DNA sequence not encode a signal sequence. If the chosen cells are eukaryotic, it generally is preferred that a signal sequence be encoded and most preferably that the wild-type IL-4 signal sequence be used.
  • Standard methods may be applied to synthesize a gene encoding an IL-4 mutein according to this invention.
  • the complete amino acid sequence may be used to construct a back-translated gene.
  • a DNA oligomer containing a nucleotide sequence coding for IL-4 mutein may be synthesized.
  • several small oligonucleotides coding for portions of the desired polypeptide may be synthesized and then ligated.
  • the individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
  • DNA sequences encoding an IL-4 mutein of this invention will be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the IL-4 mutein in the desired transformed host. Proper assembly may be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.
  • expression control sequence and expression vector will depend upon the choice of host. A wide variety of expression host/vector combinations may be employed.
  • Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus.
  • Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E.coli, including col El, pCRl, pER32z, pMB9 and their derivatives, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages.
  • Useful expression vectors for yeast cells include the 2 ⁇ plasmid and derivatives thereof.
  • Useful vectors for insect cells include pVL 941.
  • any of a wide variety of expression control sequences may be used in these vectors.
  • useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors.
  • useful expression control seguences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of phage lambda, for example PL, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., PhoA, the promoters of the yeast ⁇ -mating system, the polyhedron promotor of Baculovirus, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • Any suitable host may be used to produce the IL-4 muteins of this invention, including bacteria, fungi (including yeasts), plant, insect, mammal, or other appropriate animal cells or cell lines, as well as transgenic animals or plants. More particularly, these hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E.coli, Pseudomonas, Bacillus, Streplomyces, fungi, yeast, insect cells such as Spodoptera frugiperda (SJ9), animal cells such as Chinese hamster ovary (CHO) and mouse cells such as NS/O, African green monkey cells such as COS 1, COS 7, BSC 1 , BSC 40, and BNT
  • CHO cells and COS 7 cells in cultures and particularly the CHO cell line CHO (D HFR-).
  • vectors and expression control sequences will function equally well to express the DNA sequences described herein. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation. For example, in selecting a vector, the host must be considered because the vector must replicate in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.
  • preferred vectors for use in this invention include those that allow the DNA encoding the IL-4 muteins to be amplified in copy number. Such amplifiable vectors are well known in the art.
  • DHFR vectors able to be amplified by DHFR amplification
  • GS glutamine synthetase
  • vectors able to be amplified by DHFR amplification see, e.g., Kaufman, United States Patent 4,470,461 , Kaufman and Sharp, "Construction Of A Modular Dihydrafolate Reductase cDNA Gene: Analysis Of Signals Utilized For Efficient Expression", Mol. Cell. Biol., 2, pp. 1304-19 (1982)
  • GS glutamine synthetase
  • an expression control sequence a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the IL-4 mutein of this invention, particularly as regards potential secondary structures. Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the DNA sequences of this invention, their secretion characteristics, their ability to fold the polypeptides correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the DNA sequences.
  • IL-4 muteins obtained according to the present invention may be glycosylated or unglycosylated depending on the host organism used to produce the mutein. If bacteria are chosen as the host then the IL-4 mutein produced will be unglycosylated. Eukaryotic cells, on the other hand, will glycosylate the IL-4 muteins, although perhaps not in the same way as native IL-4 is glycosylated.
  • the IL-4 mutein produced by the transformed host can be purified according to any suitable method.
  • the biological activity of the IL-4 muteins of this invention can be assayed by any suitable method known in the art.
  • assays include antibody neutralization of antiviral activity, induction of protein kinase, oligoadenylate 2,5-A synthetase or phosphodiesterase activities, as described in EP-B1-41313.
  • assays also include immunomodulatory assays (see, e.g., United States patent 4,753,795), growth inhibition assays, T cell proliferation, induction of IL-6 (MCP-1 or VCAM-1) on EC and measurement of binding to cells that express interleukin-4 receptors.
  • the IL-4 mutein of this invention will be administered at a dose approximately paralleling that or greater than employed in therapy with wild type native or recombinant IL-4.
  • An effective amount of the IL-4 mutein is preferably administered.
  • An "effective amount” means an amount capable of preventing or lessening the severity or spread of the condition or indication being treated.
  • the effective amount of IL-4 mutein will depend, inter alia, upon the disease, the dose, the administration schedule of the IL-4 mutein, whether the IL-4 mutein is administered alone or in conjunction with other therapeutic agents, the serum half-life of the composition, and the general health of the patient.
  • the IL-4 mutein is preferably administered in a composition including a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier means a carrier that does not cause any untoward effect in patients to whom it is administered. Such pharmaceutically acceptable carriers are well known in the art. We prefer 2% HSA/PBS at pH 7.0.
  • the IL-4 muteins of the present invention can be formulated into pharmaceutical compositions by well known methods. See, e.g., Remington's Pharmacautical Science by E. W. Martin, hereby inco ⁇ orated by reference, describes suitable formulations.
  • the pharmaceutical composition of the IL-4 mutein may be formulated in a variety of forms, including liquid, gel, lyophilized, or any other suitable form. The preferred form will depend upon the particular indication being treated and will be apparent to one of skill in the art.
  • the IL-4 mutein pharmaceutical composition may be administered orally, by aerosol, intravenously, intramuscularly, intraperitoneally, intradermally or subcutaneously or in any other acceptable manner.
  • the pharmaceutical composition of the IL-4 mutein may be administered in conjunction with other therapeutic agents. These agents may be inco ⁇ orated as part of the same pharmaceutical composition or may be administered separately from the IL-4 mutein, either concurrently or in accordance with any other acceptable treatment schedule.
  • the IL-4 mutein pharmaceutical composition may be used as an adjunct to other therapies. Accordingly, this invention provides compositions and methods for treating immune disorders, cancers or tumors, abnormal cell growth, or for immunomodulation in any suitable animal, preferably a mammal, most preferably human. As previously noted in the Background section, IL-4 has many effects.
  • T cell proliferation T-helper cell differentiation
  • induction of human B-cell activation and proliferation T-helper cell activation and proliferation
  • lymphokine-directed immunoglobulin class switching Effects on the lymphoid system include increasing the expression of MHC class II antigen (Noelle, R., el al, Increased Expression of la Antigens on resting B cells: a New Role for B Cell Growth Factor, PNAS USA. 81:6149-53 (1984)), and CD 23 on B cells (Kikutani, H, et al., Molecular Structure of Human Lymphocyte Receptor for Immunoglobulin, Cell 47:657-61 (1986)).
  • T-helper cell type 1 (Thl) and type 2 (Th2) are involved in the immune response. Stimulated Th2 cells secrete IL-4 and block Thl progression. Thus, any Thl -implicated disease is amenable to treatment by IL-4 or analogs thereof.
  • Gene therapy applications contemplated include treatment of those diseases in which IL-4 is expected to provide an effective therapy due to its immunomodulatory activity, e.g., Multiple Sclerosis (MS), Insulin-dependent Diabetes Mellitus (IDDM), Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), uveitis, orchitis, primary biliary cirrhosis, malaria, leprosy, Lyme Disease, contact dermatitis, psoriasis, B cell lymphoma, acute lymphoblastic leukemia, non-Hodgkins lymphoma, cancer, osteoarthritis and diseases that are otherwise responsive to IL-4 or infectious agents sensitive to IL-4-mediated immune response.
  • MS Multiple Sclerosis
  • IDDM Insulin-dependent Diabetes Mellitus
  • RA Rheumatoid Arthritis
  • SLE Systemic Lupus Erythematosus
  • uveitis orchitis
  • IL-4 muteins may provide the therapeutic agent to the target area.
  • in vitro and in vivo gene therapy methodologies are contemplated.
  • Several methods for transferring potentially therapeutic genes to defined cell populations are known. See, e.g., Mulligan, "The Basic Science Of Gene Therapy", Science. 260: 926-31 (1993). These methods include:
  • DNA viruses include adenoviruses (preferably Ad-2 or Ad-5 based vectors), he ⁇ es viruses (preferably he ⁇ es simplex virus based vectors), and parvoviruses (preferably "defective" or non-autonomous parvovirus based vectors, more preferably adeno-associated virus based vectors, most preferably AAV-2 based vectors).
  • adenoviruses preferably Ad-2 or Ad-5 based vectors
  • he ⁇ es viruses preferably he ⁇ es simplex virus based vectors
  • parvoviruses preferably "defective" or non-autonomous parvovirus based vectors, more preferably adeno-associated virus based vectors, most preferably AAV-2 based vectors.
  • retroviral vectors have been extensively studied and used in a number of gene therapy applications, these vectors are generally unsuited for infecting non-dividing cells. In addition, retroviruses have the potential for oncogenicity.
  • Adenoviruses have the advantage that they have a broad host range, can infect quiescent or terminally differentiated cells, such as neurons or hepatocytes, and appear essentially non-oncogenic. See, e.g., AU et al, supra, p. 367. Adenoviruses do not appear to integrate into the host genome. Because they exist extrachromosomally, the risk of insertional mutagenesis is greatly reduced. AU et al, supra, p. 373.
  • Adeno-associated viruses exhibit similar advantages as adenoviral-based vectors. However, AAVs exhibit site-specific integration on human chromosome 19. AU et al, supra, p. 377.
  • the IL-4 mutein-encoding DNA of this invention is used in gene therapy for autoimmune diseases such as MS, IDDM, and RA, infectious diseases such as Lyme Disease and Leprosy, cancers, such as non-Hodgkins lymphoma and ALL, cartiledgenous disorders such as osteoarthritis, and psoriatic conditions, such as psoriasis.
  • autoimmune diseases such as MS, IDDM, and RA
  • infectious diseases such as Lyme Disease and Leprosy
  • cancers such as non-Hodgkins lymphoma and ALL
  • cartiledgenous disorders such as osteoarthritis
  • psoriatic conditions such as psoriasis.
  • gene therapy with DNA encoding the IL-4 muteins of this invention is provided to a patient in need thereof, concurrent with, or immediately after diagnosis.
  • This approach takes advantage of the selective activity of the IL-4 muteins of this invention to prevent undesired autoimmune stimulation.
  • any suitable gene therapy vector containing IL-4 mutein DNA may be used in accordance with this embodiment.
  • the techniques for constructing such a vector are known. See, e.g., Ohno et al, supra, p. 784; Chang et al, supra, p. 522.
  • Introduction of the IL-4 mutein DNA-containing vector to the target site may be accomplished using known techniques, e.g., as described in Ohno et al, supra, p. 784.
  • 125 Muteins were expressed in a baculovirus system, purified to homogeneity, and evaluated in biological assays that reflected different IL-4 receptor usage. Two assays were used to test for selective agonist activity, IL-4-induced HUVEC IL-6 secretion assay, and 1° T cell proliferation assay for positive IL-4 activity. Compounds that have the ability to induce 1 ° T cell proliferation, yet have a reduced ability to induce IL-6 secretion, are T cell selective IL-4 agonists and come within the scope of this invention. More specifically, human umbilical vein endothelial cells (HUVEC) were used to assess activity through the alternate IL-4 receptor (IL-4R ⁇ / ⁇ -/ ⁇ £e receptor component).
  • VEC human umbilical vein endothelial cells
  • Example 1 Production of muteins. Muteins were generated by site-directed mutagenesis using primers containing codons corresponding to the desired mutation essentially as described by Kunkel TA, Roberts JD, and Zakour RA, "Rapid and efficient site-specific mutagenesis without phenotypic selection” (1987), Methods Enzymol 154: 367-382. Briefly, human IL-4 cDNA containing the restricitlon enzyme sites Bam HI and Xba I was subcloned into the Ml 3 phage vector M13 mpl9 (New England Biolabs, Beverly, MA) using the same sites. Wild-type IL-4 cDNA was obtained using
  • PCR Polymerase Chain Reaction
  • Site-directed mutagenesis utilized in general primers containing 15 nucleotides homologous to the template U-DNA 5' to the codon(s) targetted for mutagnesis, nucleotides that inco ⁇ orate the desired change, and an additional 10 nucleotides homologous to the template U-DNA 3' of the last altered nucleotide.
  • the specific primers used were:
  • R121A CTAAAGACGA TCATGGCTGA GAAATATT (SEQ ID NO:24)
  • R121D GCTAAAGACG ATCATGGACG AGAAATATTC (SEQ ID NO:25)
  • R121E GCTAAAGACG ATCATGGAAG AGAAATATTC (SEQ ID NO:26)
  • R121F CTAAAGACGA TCATGTTTGA GAAATATT (SEQ ID NO:27)
  • R121H CTAAAGACGA TCATGCACGA GAAATATT (SEQ ID NO:28)
  • R121I CTAAAGACGA TCATGATAGA GAAATATT (SEQ ID NO:29)
  • R121K CTAAAGACGA TCATGAAAGA GAAATATT (SEQ ID NO:30)
  • R121N CTAAAGACGA TCATGAACGA GAAATATT (SEQ ID NO:31)
  • R121P GCTAAAGACG ATCATGCCAG AGAAATATTC (SEQ ID NO:32)
  • R121T CTAAAGACGA TCATGACTGA GAAATATT (SEQ ID NO:33)
  • R121W CTAAAGACGA TCATGTGGGA GAAATATT (SEQ ID NO:34)
  • Y124A ATCATGAGAG AGAAAGCATC AAAGTGTT (SEQ ID NO:35)
  • Y124R ATCATGAGAG AGAAACGATC AAAGTGTT (SEQ ID NO:37)
  • Y124T ATCATGAGAG AGAAAACATC AAAGTGTT (SEQ ID NO:39)
  • Y124A/S125A CGATCATGAG AGAGAAAGCT GCTAAGTGTT CGA (SEQ ID NO:40)
  • T13D CAGGAGATCA TCAAAGATTT GAACAGCC (SEQ ID NO:41)
  • R121T/E122F/Y124Q GCTAAAGACG ATCATGACCT TCAAACAGTC AAAG(SEQ ID NO:42)
  • Regions of mutated nucleotides are underlined.
  • Primers were phosphorylated using T4 polynucleotide kinase (New England Biolabs, Beverly, MA) using the manufacturer's protocol. After annealing of the primer to the U-DNA template and extension with T7 DNA polymerase (Bio-Rad Laboratories, Hercules, CA), cells of the E. coli strain DH5 ⁇ TM (GibcoBRL, Gaithersburg, MD) were transformed with 5 ⁇ l of reaction mixture and plated in LB medium containing 0.7% agar. After incubation at 37°C, plaques were expanded by picking a single plaque and transferring to 2 mis of LB media and grown overnight at 37°C.
  • Single strand DNA was isolated using an Ml 3 purification kit (Qiagen, Inc., Chatsworth, CA) per manufacturer's protocol, and clones containing the desired mutation were identified by sequencing the single stranded DNA using the Sequenase® sequencing kit (Amersham Life Sciences, Arlington Heights, IL) per manufacturer's protocol.
  • IL-4 mutein cDNA from Replicative Form DNA corresponding to plaques containing the correct mutated sequence was isolated using Bam HI and Xba I, and subcloned to the plasmid vector pFastBacTMl (GibcoBRL, Gaithersburg, MD).
  • Bacmid recombininant baculovirus DNA
  • Bacmid recombininant baculovirus DNA
  • the superaatants were harvested 48-60 hrs post-infection by centrifugation at 5000 ⁇ m for 10 minutes in a Sorvall® RC-5B centrifuge using a GSA rotor (Dupont Instrument Co., Willmington, DE) and assayed for virus titre (typically, >1X10 ⁇ plaque forming units/ml was obtained).
  • C400.1 and C400.17 were generated using standard protocols from mice using recombinant human IL-4 (Genzyme Diagnostics, Cambridge, MA) as immunogen, were produced as ascites fluid, purified, and coupled to CNBr-activated Sepharose (Pharmacia, Uppsala, Sweden) as per manufacturer's protocol.
  • Sf9 cell supernatants generated from infection of Sf 9 cells by recombinant baculovirus containing the respective IL-4 mutein were loaded onto a 1 ml column of IL-4 affinity matrix, washed with 100 mM NaHCO 3 , 500 mM NaCl, pH 8.3, washed with water to remove salt, and eluted with 8 column volumes of lOOmM Glycine, pH 3.0. Fractions were collected in siliconized vials containing 0.1 volume 1M Tris, pH 8.0.
  • Mutein protein was further purified by reverse phase chromatography using a Dynamax®-30 ⁇ A CJ S column (Rainin Instrument Co., Woburn, MA) with a 0- 100% gradient of Buffer A to B (Buffer A, water; Buffer B, acetonitrile, 0.1 % trifluoroacetic acid). Fractions were evaluated by SDS-PAGE, and mutein containing fractions were lyophilized for storage, and resuspended in sterile phosphate-buffered saline for assays. Mutein so purified was typically a single band as observed by SDS-PAGE (silver stain), and was quantitated by amino acid analysis (accuracy typically >90%).
  • Example 3 1° T cell proliferation assay.
  • Primary T cells were obtained from fresh blood from normal donors and purified by centrifugation using Ficoll-Paque® Plus (Pharmacia, Upsalla, Sweden) essentially as described by Kruse, N., Tony, H. P. and Sebald, W. "Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement", Embo J. 11: 3237-44 (1992).
  • the purified peripheral blood mononuclear cells were incubated for 7 days with 10 ⁇ g/ml phytohemagglutinin (Sigma Chemical Co., St. Louis, MO), harvested by centrifugation, and washed in RPMI 1640 media (GibcoBRL, Gaithersburg, MD).
  • HUVEC IL-6 secretion assay Human umbilical vein endothelial cells (HUVEC) were obtained from Clonetics® Corp. (San Diego, CA), and maintained as per supplier's protocols. Cells (passage 3 to 6) were harvested by incubation with Trypsin/EDTA, washed, and plated at subconfluent densities in 48-well plates in EGM® media (Clonetics® Co ⁇ ., San Diego, CA) containing bovine brain extract (BBE; Clonetics® Co ⁇ ., San Diego, CA). At confluency (3-4 days at 37°C), the media was removed and replaced with EGM ® media without BBE.
  • EGM® media Clonetics® Co ⁇ ., San Diego, CA
  • BBE bovine brain extract
  • IL-4 or mutein was added to the cells in fresh EGM® without BBE, and allowed to incubate an additional 24 hrs.
  • Supernatants were harvested and the concentration of IL-6 was analyzed using a human IL-6 ELISA. The conditions were identical except for antagonist assays, varying concentrations of mutein were added to a constant concentration of 1 OOpM IL-4. Briefly, 96-well Immunolon® 2 plates (Dynatech Laboratories, Inc., Chantilly, VA) were coated with 5 ⁇ g/ml anti-human IL-6 MAb Cat#1618-01 (Genzyme Diagnostics, Cambridge, MA) overnight at 4°C.
  • Human IL-6 standard (Genzyme Diagnostics, Cambridge, MA) or samples were titrated in duplicate and incubated with the coated plate; after washes, secondary antibody rabbit anti-human IL-6 PAb (Caltag Laboratories, South San Francisco, CA, Cat#PS-37) at a 1 : 1000 dilution was added.
  • the presence of bound rabbit anti-IL-6 PAb was detected using alkaline phosphatase-coupled donkey anti-rabbit Ig PAb (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, Cat#71 1-055-152) diluted 1 :2000, and developed using pNPP (Sigma Chemical Co.. St. Louis, MO, Cat#N2770 or N 1891). Absorbance was read at 405 nm using a VmaxTM kinetic microplate reader (Molecular Devices Co ⁇ ., Menlo Park, CA).
  • Example 5 Activities of Muteins. Table 1 summarizes the results of the muteins in the two assays described above. "EC50, pM” is the effective concentration that produces a 50% maximal response measured in the concentration picomoles/liter. Activity is a function of both potency (EC 50 ) and maximal response (R, nax ). Cell-selective muteins exhibited differential activity of either a relative reduction in R, l ⁇ ax and/or a relative reduction in potency (increase in EC 50 ) in the HUVEC assay vs. the T cell assay. "R m ax > %wt” is the maximal response measured relative to wild-type IL-4. By definition, wild-type IL-4 gives 100% response.
  • Muteins R121D, R121E, R121P, and R121T/E122F/Y124Q were more potent than wild-type IL-4 in this assay, although mutein R121T/E122F/Y124Q had a reduced maximal response .
  • Muteins Y124Q, Y124R, and Y124A/S125A had 2-3 -fold increased EC50 values than wild-type, as well as a reduced maximal response. However, they appear to retain a significant proportion of IL-4 activity on T cells.
  • Muteins R121E, Y124Q and R121T/E122F/Y124Q had no measurable activity in the HUVEC assay, making them clearly T cell-selective, and thus selective for the IL-4 receptor expressed on T cells (IL-4R ⁇ /IL-2R ⁇ ). These muteins are IL-4 antagonists on endothelial cells because, although they interact normally with IL-4R ⁇ , they do not activate the complex IL-4R ⁇ / ⁇ -/i£e subunit.
  • the muteins R121P and Y124R show activity in the HUVEC assay, but their EC50 values are increased between 50-150-fold, and have reduced maximal responses relative to their ability to stimulate T cells. Although these two proteins do not appear to be absolutely T cell-selective, they are preferential for their activation of the T cell IL-4 receptor over the HUVEC IL-4 receptor.
  • Figs. 2A and B are a series of dose-response plots of HUVEC IL-6 secretion by the T cell-selective agonists relative to wild-type.
  • IL-4 was included as an internal control on each plate used to assay mutein activity; a representative curve is shown.
  • Fig. 4A is the dose-response curve for R121E versus wild-type IL-4.
  • Fig. 2A and B are a series of dose-response plots of HUVEC IL-6 secretion by the T cell-selective agonists relative to wild-type. IL-4 was included as an internal control on each plate used to assay mutein activity; a representative curve is shown.
  • Fig. 4A is the dose-response curve for R121E versus wild-type IL-4.
  • 4B-F are the dose-response curves for R121P, Y124Q, Y124R, Y124A/S125A, and R121T/E122F/Y124Q, respectively, versus wild-type IL-4. Activities have been normalized relative to the IL-4 control responses. Muteins R121E, Y124Q, and R121T/E122F/Y124Q demonstrate no activity in this assay. Muteins R121P and Y124R, though showing partial agonist activity in this assay, are relatively less potent to wild-type IL-4 than they are in the 1° T cell assay. Thus, despite their activity, they still demonstrate preferential activation of the T cell IL-4 receptor.
  • Example 7 Biological response of IL-4 muteins in 1° T cell assays.
  • Figs. 4A and B show dose-response curves of the T cell-selective agonist muteins in a representative assay using cells from a normal donor.
  • IL-4 was included as an internal control on each plate used to assay mutein activity; a representative curve is shown.
  • Fig. 6A is the dose-response curve for R121E versus wild-type IL-4.
  • Fig. 4A and B show dose-response curves of the T cell-selective agonist muteins in a representative assay using cells from a normal donor.
  • IL-4 was included as an internal control on each plate used to assay mutein activity; a representative curve is shown.
  • Fig. 6A is the dose-response curve for R121E versus wild-type IL-4.
  • 6B-F are the dose-response curves for R121P, Y124Q, Y124R, Y124A/S125A, and R121T/E122F/Y124Q, respectively, versus wild-type IL-4. Activities have been normalized relative to the IL-4 control responses. Muteins R121E, R121P, and R121T/E122F/Y124Q are more potent than wild-type IL-4, although R121T/E122F/Y124Q is only a partial agonist in this assay.
  • muteins Y124Q, Y124R, and Y124A/S125A are still effective partial agonists (R m ax values 60-70%) of wild-type).
  • activity is relative to the IL-4 response seen in the same plate for each mutein.
  • muteins R121E and Y124Q which show significant activity in the T cell assay yet no apparent activity in the HUVEC assay.
  • Each of these muteins is a clear T cell-selective agonist.
  • Example 8 Antagonism of HUVEC IL-4-induced IL-6 secretion by T cell selective muteins.
  • the IL-4 antagonist R121D/Y124D antagonizes IL-4 with a Kj of -1.5 nM under these conditions.
  • HUVEC do not express IL-2R ⁇ , but do express the IL-4 receptor ⁇ -like subunit.
  • the substituted residues of R121E and Y124Q are in the D-helix of IL-4 and thus only affect interactions of IL-4 with IL-2R ⁇ (functional interaction) and the ⁇ -like subunit (no or non-functional interaction), but do not affect the ability of these muteins to bind to IL-4R ⁇ .
  • the T cell-selective agonists R121 E and Y124Q by virtue of their ability to selectively interact with IL-2R ⁇ on T cells without promoting activation of the ⁇ -like receptor subunit on endothelial cells, are able to compete IL-4- induced responses on these endothelial cells (Kj -0.8-1 nM under these conditions).
  • Such antagonism by T cell-selective IL-4 muteins may antagonize the effects of endogenously produced IL-4 on endothelial cells during T cell-directed therapy with said muteins.
  • Examples 9 Biological response of IL-4 mutein RI 2 II).
  • Example 10 Biological activity of IL-4 mutein T13D/R121E.
  • the IL-4 mutein T13D/R121E (Thr-13 substituted with Asp together with Arg-121 substituted with Glu) was generated as described and assayed in the 1° T cell and HUVEC assays.
  • T13D/R121E (A) exhibited an EC50 of -100 pM, about 2-3-fold better than IL-4 (O) or R121E ( ⁇ ), and an R max value of 100% relative to IL-4.
  • T13D/R121E (A) was ⁇ 27-fold more potent an antagonist of IL-4 activity than R121E ( ⁇ ), exhibiting an IC 50 of -2.2 nM vs. -60 nM, respectively.
  • the substitution of Thr- 13 to Asp increases the potency of the T13D/R121E relative to R121E (both as an agonist in the T cell assay, and as an antagonist in the HUVEC assay), while the substitution Arg-121 to Glu confers T cell-selective activity to T13D/R121E (full agonist in the T cell assay, IL-4 antagonist in the HUVEC assay).
  • Example 11 Evaluation of the IL-4 selective agonist in the pathology model
  • ketamine hydrochloride Ketaset, 10 mg/kg.
  • the upper back area of each animal is sheared and cleaned with a 70% alcohol-betadine solution.
  • IL-4, IL-4 selective agonist or vehicle (0.2% human serum albumin, HSA) is injected intradermally into the backs of animals in a volume of 0.1 ml using a 1 ml tuberculin syringe.
  • the injected sites are separated by at least 10 cm and marked with an indelible marker.
  • Tissue biopsy samples are obtained using a 6mm punch biopsy tool and samples are placed in OCT and snap frozen in liquid nitrogen. Biopsies are obtained at 0, 4, 8 and 24 hrs post injection.
  • Test article is administered subcutaneously twice daily (approximately 10-12 hr apart) over four consecutive days in a volume of 0.1 ml/kg at dosages of 0, 2.5, 25 or 250 ug/kg resulting in a total daily dose of 0, 5, 50 and 500 ug/kg, respectively.
  • a peripheral blood sample is obtained from each animal prior to the first injection of vehicle or IL-4 and at the beginning of each day of the study, and aliquots analyzed for complete blood cell counts and differentials, and flow cytometry analysis of peripheral blood mononuclear cell surface markers. The remainder of the blood sample is centrifuged and plasma aliquots stored at -70° C for subsequent analysis of chemokine levels.
  • Frozen sections are prepared and allowed to equilibrate to room temperature, air dried and fixed in acetone at 4 C for 5 minutes. Slides are transferred to 10 mM PBS with 0.1% BSA for 5 minutes. VCAM-1 is localized with the use of C313.3, a monoclonal antibody to human VCAM-1. An irrelevant, i so type-matched immunoglobulin at the appropriate concentration is used as a negative control. Endogenous biotin is blocked using the Vector Biotin blocking kit (Vector Laboratories, Burlingame, CA). Sections are incubated 1.5 hr at room temperature in a humid chamber with the indicated antisera diluted in PBS with 0.1% BSA and 1% normal rabbit serum.
  • Vector Biotin blocking kit Vector Biotin blocking kit
  • the slides are stained with the Vector ABC Elite kit according to manufacturers directions and the antibody conjugate detected by incubating the slides in 3-amino-9- ethylcarbazole/hydrogen peroxide (AEC substrate kit, Vector). Sections are washed thoroughly in 0.1M acetate buffer, washed in distilled water and mounted in Lerner AQUA-MOUNT (Lerner Laboratories, Pittsburgh, PA).
  • Specimens are scored by two independent observers in a blinded manner using set scales between 0 and 3+, designed to assess the intensity as well as distribution of staining.
  • the scoring system for VCAM-1 expression is: 0 absent or faint staining of occasional vessel; 1+ faint staining of several vessels; 2+ moderate intensity staining of most vessels; 3+ intense staining of most vessels.
  • Blood vessels are identified in serial sections stained for von Willebrands Factor (polyclonal rabbit anti-human VWF; Dakoplatts, Ca ⁇ interia, CA).
  • erythrocyte count, hematocrit, leukocyte count and platelet counts are performed on heparinized blood samples with a Serono 9000 Blood Analyzer (Baker Diagnostics, Allcntown, PA).
  • Leukocyte differentials are evaluated on Diff-Quick stained blood smears where a total of two hundred cells are counted and the percentage of each cell type was recorded.
  • PBMC surface markers Analysis of peripheral blood mononuclear cell (PBMC) surface markers is performed in the following manner.
  • a 4 ml sample of heparinized blood is diluted in Hanks Balanced Salt Solution (HBSS, without Mg++ or Ca++) and layered onto 4 ml of Percoll (1.070 gm/ml density).
  • the tubes are centrifuged at 1800 ⁇ m (Beckman GS-6R) for 20 minutes at 24 C.
  • the lymphocyte containing layer is aspirated and centrifuged at 1 100 ⁇ m for 10 minutes.
  • the resultant cell pellet is resuspended in 6 ml of phosphate buffered saline (PBS) containing 0.1% Azide and 5% goat serum. Aliquots of 1 ml are utilized for cell surface marker analysis as described below.
  • PBS phosphate buffered saline
  • Antibodies against CD2, CD4, CD8, CD1 lb,.CD16, CD25, CD49 and HLA-DR are utilized for analysis by flow cytometry. Twenty ul aliquots of marker antibodies are incubated with 1 ml aliquots of cell suspension in the dark for 60 minutes at 4 C, and samples centrifuged (1000 ⁇ m, 10 min at 4 C). The pellets are washed three times with 1 ml of PBS containing 0.1 % azide and 5% goat serum, followed by FACs analysis. Plasma samples obtained during each study are analyzed for levels of MCP-1 by specific ELISA.
  • 96 well plates (Nunc, Kamstrup, Denmark) are coated with 50 ul/well rabbit anti-MCP-1 for 16 hr at 4 C and then washed in PBS, pH 7.5, 0.05% Tween-20 (wash buffer). Non specific binding sites are blocked with 2% BSA in PBS (200 ul) and the plates incubated for 90 minutes at 37 C. Plates are rinsed three times with wash buffer, and diluted (neat, 1 :5 and 1 :10) test sample (50 ul) in duplicate is added, followed by incubation for 1 hr at 37 C. Plates are washed four times, and 50 ul/well biotinylated rabbit anti-MCP-1 is added for 45 minutes at 37 C.
  • Plates are washed four times, streptavidin-peroxidase conjugate (100 ug/ml) (Dakopatts, Ca ⁇ interia, CA) is added and the plates are incubated for 30 minutes at 37 C. The plates are washed three times, and 100 ul chromogen substrate (0.67 mg/ml orthophenylenediamine dichloride (Dakopatts, Ca ⁇ interia, CA) is added. The plates are incubated at 25 C for 6 minutes and the reaction is terminated with 50 ul/well of 3 M H2SO4 solution in wash buffer plus 2% FCS. Plates are read at 490 nm in an ELISA reader. Standards are 0.5 log dilutions of recombinant MCP-1 from 100 ng/ml to 1 pg/ml (50 ul/well). The ELISA consistently detects MCP-1 concentrations > 50 pg/ml.
  • Example 12 Treatment of multiple sclerosis with IL-4 selective agonist
  • MS multiple sclerosis
  • EAE Experimental autoimmune encephalomyelitis
  • C. jacchus marmosets
  • CFA complete Freund's adjuvant
  • EAE EAE is assessed by clinical and pathological criteria.
  • Magnetic resonance imaging has been shown to be a useful technique to characterize early as well as late immune mediated lesions of MS (Stewart et al, Brain, 114: 1069 (1991 ).
  • MRI is used to evaluate animals after immunization to monitor progression of disease over time.
  • MRI data is collected on a Picker International NMR Cryogenic '2000' system, operating at a field strength of 0.15 Tesla; a receiver coil with an aperture of 15 cm to obtain the images.
  • Multislice spin-echo and inversion-recovery pulse sequences are employed. Echo-delays times of either 40 and 60 ms, or 40 and 80 ms are used in the spin-echo sequences.
  • the 180 -90 inte ⁇ ulse delay is 400 ms.
  • Marmosets are anesthetized with ketamine hydrochloride and placed in the scanner using a laser available for patient alignment such that the inner canthi of the eyes are aligned pe ⁇ endicular to the direction of the static magnetic field.
  • Animals are scanned before immunization and then daily from day 9 after immunization. Prior to scanning each day, animals are checked for signs of neurological impairment. Animals are sacrificed at different times after immunization. The CNS is removed and fixed in 10% formalin. Paraffin sections of brain and spinal cord are prepared and stained with hematoxylin and eosin.
  • test drug is administered subcutaneously at a dosage range between 1 and 500 ug/kg following a dosing regimen of 1 administration per day to 1 administration per week prior to the onset of disease symptoms.
  • test article is administered subcutaneously at a dose range between 1 and 500 ug/kg following an extended dosing regimen of 1 treatment per day to 1 treatment per week over the course of several months.
  • Example 13 Treatment of rheumatoid arthritis
  • RA Rheumatoid arthritis
  • PBS phosphate buffered saline
  • tissue is obtained for pathological and histopathology examination.
  • the tissue is processed for immunohistochemical staining (frozen sections) or fixed and embedded in paraffin, sectioned and stained with H&E for analysis of cellular infiltration.
  • a murine analog of the IL-4 selective agonist of the present invention in the CIA model is performed with the use of a murine equivalent protein molecule.
  • One of ordinary skill in the art is capable of comparing the murine IL-4 structure with the human IL-4 structure, generating parallel murine IL-4 muteins, and making any necessary adjustments based on responses in in vitro assays utilizing cell lines expressing either IL-4R ⁇ /IL-2R ⁇ or IL-4R ⁇ / ⁇ -like subunit in a manner analogous to that used for human IL-4 muteins with T cells and HUVEC.
  • Animals are dosed one day prior to the booster administration of collagen and kept on a dosing regimen ranging between once a day to once a week for the duration of the study (40+ days). Animals are dosed with a range of concentrations of IL-4 selective agonist ranging between 1 to 100 ug/kg.
  • Example 14 Treatment of insulin dependent diabetes mellitus (IDDM) There is some evidence in the literature of Thl cell involvement in IDDM in humans and animal models of human disease. Nonobese diabetic (NOD) mice are utilized to examine the efficacy of a murine IL-4 equivalent of IL-4 selective agonist in treating IDDM.
  • NOD nonobese diabetic mice
  • One of ordinary skill in the art is capable of comparing the murine IL-4 structure with the human IL-4 structure, generating parallel murine IL-4 muteins, and making any necessary adjustments based on responses in in vitro assays utilizing cell lines expressing either IL-4R ⁇ /IL-2R ⁇ or IL-4R ⁇ / ⁇ -like subunit in a manner analogous to that used for human IL-4 muteins with T cells and HUVEC.
  • Prediabetic NOD mice (approximately 7 wks) exhibit a proliferative unresponsiveness in vitro after T cell stimulation. The timing of this unresponsiveness is not related to insulitis and persists until the onset of diabetes which occurs at 24 wks of age. Evaluation of the IL-4 selective agonist in NOD mice is conducted similar to studies reported by Rapoport et al., J. Exp Med; 178; p. 87 (1993). NOD mice are injected with test material at approximately 3 wks of age following a dosing regimen of once daily treatment or once a week treatment over the course of 12 weeks until the mice are 15 wks old. A control group of animals will receive treatment with a inert protein equivalent.
  • mice will we tested for glycosuria using Tes-Tape and diagnosed for diabetes as determined by being glycosuria for at least two consecutive weeks.
  • animals are sacrificed to obtain various organs and tissue for pathology evaluation. Tissue from the pancreas, submandibular salivary glands and kidney from each mouse is fixed and embedded in paraffin, sectioned and stained. Aldehyde fuchsin staining of pancreas sections is used to examine the extent to which insulitic infiltrates have reduced the mass of granulated ⁇ cells.
  • Splenic leukocytes are counted by FACScan analyses using anti-Thy-1.2, anti-CD4 and anti-CD8 mabs in ascites as described by Zipris et al., J. Immunol 146; p. 3763 (1991).
  • SEQ ID NO: 1 hIL-4 (amino acid)
  • SEQ ID NO: 2 hIL-4 (amino acid, cDNA)
  • SEQ ID NO: 3 R121A (amino acid, cDNA)
  • SEQ ID NO: 4 R121D (amino acid, cDNA)
  • SEQ ID NO: 5 R121E (amino acid, cDNA)
  • SEQ ID NO: 6 R121F (amino acid, cDNA)
  • SEQ ID NO: 7 R121H (amino acid, cDNA)
  • SEQ ID NO: 8 R121I (amino acid, cDNA)
  • SEQ ID NO: 9 R121K (amino acid, cDNA)
  • SEQ ID NO: 10 R121N (amino acid, DNA)
  • SEQ ID NO: 1 1 R121P (amino acid, cDNA)
  • SEQ ID NO: 12 R121T (amino acid, cDNA)
  • SEQ ID NO: 13 R121 W (amino acid, cDNA)
  • SEQ ID NO: 14 Y124A (amino acid, cDNA)
  • SEQ ID NO: 15 Y124Q (amino acid, cDNA)
  • SEQ ID NO: 16 Y124R (amino acid, cDNA)
  • SEQ ID NO: 17 Y121 S (amino acid, cDNA)
  • SEQ ID NO: 18 R121T (amino acid, cDNA)
  • SEQ ID NO: 19 Y124A/S125A (amino acid, cDNA)
  • SEQ ID NO: 20 T13D/R121E (amino acid, cDNA)
  • SEQ ID NO: 21 R121T/E122F/Y124Q (amino acid, cDNA)
  • SEQ ID NO: 24 Mutagenesis Primer for R121A
  • SEQ ID NO: 25 Mutagenesis Primer for R121D
  • SEQ ID NO: 26 Mutagenesis Primer for R121E
  • SEQ ID NO: 27 Mutagenesis Primer for R121F
  • SEQ ID NO: 28 Mutagenesis Primer for R121H
  • SEQ ID NO: 29 Mutagenesis Primer for R121I
  • SEQ ID NO: 30 Mutagenesis Primer for R121K
  • SEQ ID NO: 31 Mutagenesis Primer for R121N
  • SEQ ID NO: 32 Mutagenesis Primer for R121P
  • SEQ ID NO: 33 Mutagenesis Primer for R121T
  • SEQ ID NO: 34 Mutagenesis Primer for R121 W
  • SEQ ID NO: 35 Mutagenesis Primer for Y124A
  • SEQ ID NO: 36 Mutagenesis Primer for Y124Q
  • SEQ ID NO: 37 Mutagenesis Primer for Y124R
  • SEQ ID NO: 38 Mutagenesis Primer for Y124S
  • SEQ ID NO: 39 Mutagenesis Primer for Y124T
  • SEQ ID NO: 40 Mutagenesis Primer for Y124A/S125A
  • SEQ ID NO: 41 Mutagenesis Primer for T13D
  • SEQ ID NO: 42 Mutagenesis Primer for R121T/E122F/Y124Q Note: for the T13D/R121E mutein, the primers SEQ ID NOs: 26 and 41 are used.
  • AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AGO GCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala Ala Thr Val 65 70 75

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PL97330413A PL187846B1 (pl) 1996-06-14 1997-05-30 Agoniści interleukiny-4 wybiórczy dla limfocytów T
AU32220/97A AU725090B2 (en) 1996-06-14 1997-05-30 T-cell selective interleukin-4 agonists
CA002256459A CA2256459A1 (en) 1996-06-14 1997-05-30 T-cell selective interleukin-4 agonists
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EP0939817B1 (en) * 1996-07-19 2005-11-23 Bayer Corporation High-affinity interleukin-4 muteins
WO2008003514A2 (en) * 2006-07-06 2008-01-10 Apogenix Gmbh Human il-4 muteins in combination with chemotherapeutics or pro-apoptotic agents in cancer therapy
EP1893235A2 (en) * 2005-06-17 2008-03-05 Aerovance, Inc. Methods for treating dermatitis using mutant human il-4 compositions
US7947648B2 (en) * 2006-01-11 2011-05-24 Aerovance, Inc. Methods for treating asthma in human and non human primates using IL-4 mutant compositions
EP2596802A1 (en) 2011-11-23 2013-05-29 PLS-Design GmbH Pharmaceutical composition for treatment of allergic reactions
WO2017001568A1 (en) 2015-07-01 2017-01-05 Universitat De Lleida Treatment and prevention of amyotrophic lateral sclerosis

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BRPI0818287A2 (pt) * 2007-10-08 2015-11-03 Intrexon Corp células dendríticas manipuladas e usos para o tratamento do câncer.

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WO1993021308A1 (en) * 1992-04-17 1993-10-28 The University Hospital Mutant cytokines having increased receptor affinity

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KRUSE, N. ET AL.: "Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement" EMBO JOURNAL, vol. 11, no. 9, 9 September 1992, pages 3237-3244, XP002045478 cited in the application *
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0939817B1 (en) * 1996-07-19 2005-11-23 Bayer Corporation High-affinity interleukin-4 muteins
EP1893235A2 (en) * 2005-06-17 2008-03-05 Aerovance, Inc. Methods for treating dermatitis using mutant human il-4 compositions
EP1893235A4 (en) * 2005-06-17 2009-09-23 Aerovance Inc METHODS OF TREATING DERMATITIS USING MUTANT HUMAN IL-4 COMPOSITIONS
US7947648B2 (en) * 2006-01-11 2011-05-24 Aerovance, Inc. Methods for treating asthma in human and non human primates using IL-4 mutant compositions
WO2008003514A2 (en) * 2006-07-06 2008-01-10 Apogenix Gmbh Human il-4 muteins in combination with chemotherapeutics or pro-apoptotic agents in cancer therapy
WO2008003514A3 (en) * 2006-07-06 2008-06-12 Apogenix Gmbh Human il-4 muteins in combination with chemotherapeutics or pro-apoptotic agents in cancer therapy
EP2596802A1 (en) 2011-11-23 2013-05-29 PLS-Design GmbH Pharmaceutical composition for treatment of allergic reactions
WO2013075846A1 (en) 2011-11-23 2013-05-30 Pls Design Gmbh Pharmaceutical composition for treatment of allergic reactions
WO2017001568A1 (en) 2015-07-01 2017-01-05 Universitat De Lleida Treatment and prevention of amyotrophic lateral sclerosis

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